The broad goal of this proposal is to understand, and take advantage of, metabolic changes that are involved in cancer cell death induced by the common diabetic drug metformin. Metformin treatment of cancer cells leads to accumulation of dysfunctional mitochondria which is associated with cells death. Our new preliminary data show that metformin causes hexokinase II (HKII) to dissociate from mitochondria and promotes depletion of cellular ATP and NAD+. These events, as well as metformin-mediated cell death, are strongly enhanced by glucose deprivation. This is not observed in non-transformed cells. NAD+ depletion following metformin treatment also appears to be associated with changes in protein acetylation. Finally, addition of exogenous NAD+ or overexpression of NAMPT, the rate limiting enzyme in NAD synthesis, protects cells against metformin cytotoxicity. Based on these findings, we hypothesize that metformin-mediated cancer cell death is associated with depletion of ATP and NAD+ and specific effects on a key glycolytic enzyme, HKII, and on NAD-dependent sirtuin protein deacetylase pathways. This hypothesis will be tested through two specific aims. Aim 1 is to dissect the role of glucose and glycolysis on metformin-mediated cell death of cancer cells, to determine the significance of HKII dissociation from mitochondria, and to establish a mouse model to examine the interaction between glucose levels and metformin in treating cancer. Genetic approaches will be used to alter expression of HKII and then the affect on metformin cytotoxicity will be measured. The importance of mitochondrial association by HKII will be examined by expressing deletion constructs that lack the mitochondrial binding domain or by using peptides and compounds that are known to disrupt HKII binding to mitochondria. A mouse model will be developed using a carbohydrate-restricted ketogenic diet to reduce glucose availability to determine if this enhances metformin's anti-tumor activity in vivo. Also metformin will be combined with drugs that target hexokinase activity or localization to determine if this improves the anti-tumor effects. Aim 2 is to determine how NAD+ and NAMPT protect cells against metformin cytotoxicity. We will examine the effects of NAMPT overexpression, or inhibition, on metformin-mediated changes in energy metabolism and cell killing. We will determine if the inhibition of specific sirtuin deacetylases is involved in metforin-mediated changes in metabolism and cell survival. We will identify acetylated proteins that change in abundance upon metformin treatment of cancer cells. We will use mouse models to examine the effects of NAMPT expression and NAD+ precursors on metformin inhibition of tumor growth. We will determine the potential for inhibitors of NAMPT and sirtuin deacetylases to potentiate the action of metformin against tumor growth. With the completion of this work we will have a more complete understanding of the molecular mechanism of action of metformin on cancer cells. We will have an improved rationale for re-purposing of metformin for cancer therapy and we will have new insights on how to improve the efficacy of the drug.